Decreased plasma ELABELA level as a novel screening indicator for heart failure: a cohort and observational study

The predictive power of B-type natriuretic peptide (BNP) and left ventricular ejection fraction (LVEF) is limited by its low specificity in patients with heart failure (HF). Discovery of more novel biomarkers for HF better diagnosis is necessary and urgent. ELABELA, an early endogenous ligand for the G protein-coupled receptor APJ (Apelin peptide jejunum, Apelin receptor), exhibits cardioprotective actions. However, the relationship between plasma ELABELA and cardiac function in HF patients is unclear. To evaluate plasma ELABELA level and its diagnostic value in HF patients, a total of 335 patients with or without HF were recruited for our monocentric observational study. Plasma ELABELA and Apelin levels were detected by immunoassay in all patients. Spearman correlation analysis was used to analyze the correlation between plasma ELABELA or Apelin levels and study variables. The receiver operating characteristic curves were used to access the predictive power of plasma ELABELA or Apelin levels. Plasma ELABELA levels were lower, while plasma Apelin levels were higher in HF patients than in non-HF patients. Plasma ELABELA levels were gradually decreased with increasing New York Heart Association grade or decreasing LVEF. Plasma ELABELA levels were negatively correlated with BNP, left atrial diameter, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, and left ventricular posterior wall thickness and positively correlated with LVEF in HF patients. In contrast, the correlation between plasma Apelin levels and these parameters is utterly opposite to ELABELA. The diagnostic value of ELABELA, Apelin, and LVEF for all HF patients was 0.835, 0.673, and 0.612; the sensitivity was 62.52, 66.20, and 32.97%; and the specificity was 95.92, 67.23, and 87.49%, respectively. All these parameters in HF patients with preserved ejection fraction were comparable to those in total HF patients. Overall, plasma ELABELA levels were significantly reduced and negatively correlated with cardiac function in HF patients. Decreased plasma ELABELA levels may function as a novel screening biomarker for HF. A combined assessment of BNP and ELABELA may be a good choice to increase the accuracy of the diagnosis of HF.

A combined assessment of BNP and other factors can improve HF diagnosis 8 .Thus, it is necessary and urgent to discover more novel biomarkers for HF diagnosis.
The Apelinergic system consists of a G protein-coupled receptor APJ (Apelin peptide jejunum, Apelin receptor, encoded by Aplnr) 9 and two endogenous peptide ligands ELABELA (encoded by Apela, also called Toddler) 10,11 and Apelin (encoded by Apln) 12 .The Apelinergic system significantly regulates cardiovascular homeostasis and functions as a potential therapeutic target of cardiovascular diseases 13,14 .Notably, the Apelin peptide could ameliorate acute HF by inhibiting endoplasmic reticulum stress 15 , ELABELA could protect against hypertensive-induced cardiac damage by inhibiting FoxM1/ACE signaling 16 and improve left ventricular filling in cecal ligation puncture rats 17 .These results indicated the participation of Apelin and ELABELA in preventing HF.However, studies have indicated that ELABELA may be more efficient than Apelin 18 .
Accumulating evidence showed that Apelin and ELABELA exert similar important bioeffects, including cardiorenal protective action, anti-hypertension action, and positive inotropic effect 14,18,19 .As reported, the status of the plasma Apelin levels in HF patients is still controversial.Many studies have demonstrated decreased plasma Apelin levels [20][21][22][23][24][25][26] , while several other studies have indicated increased or unchanged plasma Apelin levels in HF patients [27][28][29][30] .Similarly, plasma ELABELA levels were markedly elevated in patients with myocardial infarction 31,32 and complete atrioventricular block 33 but reduced significantly in patients with congenital heart disease 34 and atrial fibrillation 35,36 .Plasma ELABELA levels were also decreased in patients with hypertension 37 and renal impairment 38 .Of note, hypertension and renal impairment are two independent risk factors for HF progression 39,40 , implying the potential biomarker function of ELABELA for HF.However, only one small cohort study indirectly demonstrated the correlation between plasma ELABELA and cardiac function in HF patients 41 .The levels of plasma ELABELA were significantly decreased in hypertensive patients with HF compared to those in hypertensive patients without HF 41 .In the present study, with the maximum exclusion of interference from other complications, we further evaluated the plasma ELABELA and Apelin levels and investigate the association between plasma ELABELA or Apelin levels and cardiac function in HF patients.Additionally, we also compared the diagnostic value of ELABELA, Apelin, and LVEF in HF patients.

Study population
The studies involving human participants were reviewed and approved by the Ethics Committee of the Affiliated Hospital of the Jiangxi University of Chinese Medicine (JZFYLL20230208002) and performed in accordance with the World Medical Association Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects.All individual patients/participants provided their written informed consent and clinical characteristics to participate in this study at enrollment.All subjects were recruited in the Department of Cardiology, Affiliated Hospital of Jiangxi University of Chinese Medicine, between December 2022 and July 2023 and divided into Non-HF and HF groups.All the laboratory assessments, except plasma ELABELA and Apelin levels, were conducted in the clinical laboratory center according to the standard protocols.Inclusion criteria for diagnosis and classification of HF, including (1) typical signs (i.e., dyspnea), (2) typical symptoms (i.e., pulmonary rales), (3) increased plasma brain natriuretic peptide (BNP) concentrations [> 95 pg/ml according to the New York Heart Association (NYHA) functional classification.],(4) ultrasound cardiogram report (impaired cardiac function evaluated by echocardiography), and (5) X-ray examination (i.e., enlarged heart shadow) were based on the 2021 ESC Guidelines for the diagnosis and treatment of HF 5 .Heart failure with preserved EF (HFpEF), midrange EF (HFmrEF), and reduced ejection fraction (HFrEF) was defined as EF ≥ 50%, > 40 but < 50, and ≤ 40%, respectively 5 .The exclusion criteria were: (1) heart diseases (i.e., atrial fibrillation, congenital heart disease, acute myocardial infarction, cardiomyopathy), (2) severe renal dysfunction (grade 4 or higher), (3) malignant tumor, (4) inferior airway diseases (i.e., acute pulmonary embolism, severe disease of lung parenchyma), (5) autoimmune related diseases (i.e., severe infection, autoimmune disease, autoimmune deficiency disease), and (6) acute stroke.In this regard, on the premise of following the inclusion and exclusion criteria, patients from the Department of Cardiology with plasma BNP concentrations ≥ 95 pg/ml were included in the HF group and patients without symptoms and signs of HF with plasma BNP concentrations < 95 pg/ml were included in the Non-HF group.All of the patients/participants did not receive the optimized treatment before collecting blood samples.

ELISA assays for plasma ELABELA and Apelin
All the fasting blood samples were collected from a peripheral vein of all patients within 24 h of admission.Upon collection, blood samples were immediately centrifuged for 5 min at 4 °C and 3000 rpm to separate plasma.The plasma samples were stored at − 80 °C for ELABELA and Apelin analysis by ELISA using the commercialized human ELABELA ELISA Kit (S-1508, Peninsula Laboratories International, Inc. USA) and Apelin ELISA kit (E01T0015, Bluegene Tech Inc., Shanghai, China) according to the manufacturers' instructions, respectively.For the measurement of plasma ELABELA, the plasma samples were appropriately extracted.

Statistical analysis
Continuous data were expressed as mean ± standard deviation (SD) for normally distributed data, median and interquartile range (IQR) for non-normally distributed data, and categorical variables as number and percentage.Student's t-test was used for intergroup differences in continuous normally distributed variables between two groups, one-way analysis of variance followed by Bonferroni comparisons and unpaired tests was used for continuous normally distributed variables among more than two groups.Spearman correlation analysis was used to correlate plasma ELABELA or Apelin levels and study variables.The clinical characteristics associated with HF were analyzed through univariate and multivariate logistic regression in all subjects.The diagnostic value of plasma ELABELA, Apelin, and LVEF was assessed by determining the area under the receiver operating

New and noteworthy
The predictive power of BNP and LVEF is limited by its low specificity in patients with HF.We reported that plasma ELABELA was significantly reduced and negatively correlated with cardiac function in HF patients, utterly opposite to the changes in plasma BNP and Apelin.Plasma ELABELA might be superior to Apelin and LVEF for the diagnosis and prognosis of HF, at least in patients with HFpEF.Combined assessment of BNP and ELABELA may provide potential benefits for the diagnosis of HF.

The baseline characteristics of patients
A total of 335 participants with and without HF were enrolled in the Non-HF group (n = 119, 68.4 ± 12.2 years) and the HF group (n = 216, 69.6 ± 11.6 years, p = 0.176 vs. Non-HF group), respectively; the other baseline clinical characteristics are shown in Table 1.There were no significant differences in sex, body mass index (BMI), comorbidities, or blood pressure between the two groups.For complications, the incidence of coronary heart disease was higher in the HF than in the Non-HF group (22.2 vs. 16.8%,p = 0.047).Data from laboratory examinations revealed that plasma BNP, creatinine, urea nitrogen, uric acid, and low-density lipoprotein cholesterol (LDL-c) levels were all higher in the HF group compared to the non-HF group (P < 0.05).In contrast, the plasma total cholesterol and triglyceride levels were lower in the HF group compared to the non-HF group (P < 0.05).Compared to the non-HF group, the HF group showed lower left ventricular ejection fraction (LVEF) and higher left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), and left ventricular posterior wall (LVPW) thickness (p < 0.05), with no significant difference in interventricular septum thickness (IVST) and right ventricular internal dimension diameter (RVIDd) between non-HF and HF groups.

Plasma BNP levels in patients with HF and without HF
Plasma BNP levels were higher in HF patients than in non-HF groups (Table 1).We further divided the 216 HF patients into four subgroups [NYHA I (57/216), II (32/216), III (39/216), and IV (88/216)] according to the classification of NYHA, or HFpEF (191/216), HFmrEF (15/216), and HFrEF (10/216) groups determined by LVEF.Plasma BNP levels were further increased with severity stratified by NYHA grade (Table 2) and the continuous reduction of LVEF (Table 3), and a similar gradient reduction of LVEF was observed in patients in both classification models.

Plasma Apelin levels in patients with HF and without HF
Plasma Apelin levels were higher in HF patients than in non-HF groups (116.5 ± 16.8 vs. 27.2 ± 11.3 ng/ml, p < 0.001, Fig. 1B) and in HF patients with NYHA II, III, and IV grade than in non-HF groups with no significant difference between non-HF and NYHA I patients (Table 2).The severity stratified by NYHA grade (Table 2) or LVEF levels (Table 3) did not affect the plasma levels of Apelin in HF patients.

Discussion
The present study aimed to investigate the potential diagnostic value of plasma ELABELA in HF patients compared to LVEF and Apelin.Our data have demonstrated that plasma ELABELA was significantly reduced and correlated with increasing NYHA grade or decreasing LVEF, utterly opposite to the changes in plasma BNP.On the contrary, plasma Apelin was significantly elevated but was not affected by the severity stratified by NYHA grade, the reduction of LVEF, or the sustained increase of plasma BNP in HF patients.We found that the levels of plasma ELABELA were negatively associated with LAD, LVEDd, LVEDs, LVPW, and plasma BNP and positively correlated with LVEF.In contrast, the correlation between plasma Apelin levels and these parameters was utterly opposite to that of plasma ELABELA.Moreover, although the differences in sensitivity between ELABELA and Apelin or LVEF for the diagnosis of HF depend on the specific NYHA grade, the diagnostic values and specificity of plasma ELABELA level for HF were higher than those of plasma Apelin and LVEF.Thus, reduced plasma ELABELA level may be a novel promising diagnostic indicator for HF patients.These findings may guide the use of ELABELA as a screening indicator for HF patients and call for further preclinical and clinical evaluation of the cardioprotective property of ELABELA in the setting of HF and the underlying mechanisms.
Although several studies have demonstrated an increase in plasma Apelin levels in NYHA II and III HF patients 27,42 , some other studies have reported a significant downregulation in plasma Apelin levels in HF 21,22,24,43 and atrial fibrillation (AF) 44 patients.Here we also found an elevated plasma Apelin level in HF patients.The reasons for these discrepancies are unclear but could be related to the difference in the study population and/or the testing methods (especially the differences in Apelin ELISA kits).Nevertheless, these studies implied a close correlation between plasma Apelin and the pathogenesis of HF.Indeed, Apelin exhibited a cardioprotective action in dilated cardiomyopathy 45 and HF 15,46 in animals.Due to the similar bioeffects with Apelin, ELABELA also exhibited similar cardiovascular-protective actions 18,19 .The current study demonstrated that plasma ELABELA levels were significantly reduced with the severity stratified by NYHA grade or decreasing LVEF in HF patients compared with non-HF patients.Although a previous study by Ma et al. has demonstrated the declined plasma ELABELA levels in hypertensive patients with HF 41 , preliminary indicating the status of plasma ELABELA in HF patients.However, they cannot rule out the interference from other complications, including hypertension and renal impairment.What's more, previous reports have already shown that plasma ELABELA concentrations were increased in patients with coronary heart diseases [31][32][33] but reduced in patients with congenital heart disease 34 , renal impairment 38 , hypertension 37 , and AF 35 , indicating the independent impact of these complications on plasma ELABELA levels.As an extension, our study excluded the influence of the above complications and demonstrated the association of the declined plasma ELABELA levels with a high risk of HF progression.In this regard, patients with lower circulating ELABELA exhibited more severe cardiac dysfunction than those with higher circulating ELABELA.In addition, our study also showed that ELABELA was also positively related to, while Apelin had a negative correlation with (no significant statistical significance) plasma LDL-c, HDL-c, cholesterol, and triglyceride.These results indicated that ELABELA might be similar to Apelin in participating in metabolic regulation or metabolic related diseases such as atherosclerosis [47][48][49] .Recently, a small cohort study has shown that plasma ELABELA levels were negatively associated with carotid intima-media thickness in hypertensive patients, indicating the potential involvement of reduced ELABELA in the pathogenesis of hypertension-associated subclinical atherosclerosis 50 .It is important to note that atherosclerosis is considered to be the main cause of most cardiovascular diseases worldwide, with ischemic heart disease as its main clinical manifestation 51 .Thus, these pieces of evidence further support the potential importance of the declined circulating ELABELA in HF pathophysiology.However, the relationship between ELABELA and the progression of atherosclerosis needs to be further clarified.We further compared plasma levels of ELABELA versus Apelin in different types of HF.Interestingly, plasma levels of ELABELA gradually decreased with increasing NYHA grade.In contrast, plasma Apelin levels were initially elevated in NYHA II patients compared to non-HF and NYHA I patients and then slightly decreased in NYHA III and IV patients compared to NYHA II patients with no statistical significance.Similarly, plasma ELABELA levels in the HF patients with HFpEF, HFrEF, or HFmrEF were sequentially decreased and lower than that in the non-HF patients.In contrary, The HF patients with HFpEF, HFrEF, or HFmrEF had higher plasma Apelin levels than non-HF patients.Interestingly, neither plasma Apelin levels between the HFpEF and HFmrEF or HFrEF group nor between HFmrEF and HFrEF group showed notable differences.These results indicated that decreased ELABELA level is more closely associated with impaired left ventricular systolic function than Apelin.There is no relationship between plasma Apelin levels and cardiac function.Notably, worsened heart function has been known as an independent risk factor for adverse events in HF patients 52 .Thus, the reduced plasma ELABELA levels rather than elevated plasma Apelin levels may be connected to adverse events in HF patients.Although studies have reported the positive inotropic effects and anti-myocardial fibrosis actions of ELABELA and Apelin 14,16,17,53 , ELABELA exhibited more effectively improving left ventricular filling in rats with cecal ligation puncture-induced sepsis 17 , achieve pronounced effects on cardiac contractions 54 , and reduce blood pressure and improve cardiorenal dysfunctions in spontaneously hypertensive rats 55 .The positive inotropic effect of Apelin was not matched to the elevated plasma Apelin levels in patients with HF.The reasons for this are not yet clear.However, we speculate that the increased plasma Apelin levels may be a compensation to the reduced plasma ELABELA levels and contribute to the alleviation of HF.This is supported by the observation that plasma Apelin levels were negatively correlated to plasma ELABELA levels in HF patients in our study.
HFpEF is recognized as a heterogeneous clinical syndrome and accounts for at least 50% of all HF patients 56 .However, the diagnosis of HFpEF is still challenging.In this regard, the European Society of (ESC) has offered a complex definition of HFpEF which includes the symptoms and signs of HF, with evidence of structural and/or functional cardiac abnormalities and/or raised natriuretic peptides, and with an LVEF more than 50% 57 .Similarly, clinicians in clinical practice have adopted another definition of HFpEF which includes an LVEF more than 40%, an elevated N-terminal pro-BNP (NT-proBNP) level, and a structural cardiac abnormality on echocardiography 57 .Of note, approximately 20% of HFpEF patients with have normal natriuretic peptide levels 58 and guidelines use the combination of LVEF ≥ 50% with functional abnormality assessment with tissue Doppler imaging to diagnose HFpEF 57 .Thus, the diagnosis of HFpEF is more difficult than that of HFmrEF or HFrEF and there is still no simpler definition specifying the use of a combination of imaging or natriuretic peptides in the diagnosis of HFpEF.In the present study, the great majority of the HF patients were diagnosed with HFpEF (88.43%), the percentages of the HFmrEF and HFrEF patients were only 6.94% and 4.62%, respectively.The diagnostic value and specificity of plasma ELABELA in patients with HFpEF were comparable with that in All HF patients and significantly higher than those of Apelin or LVEF.This may suggest the diagnostic potential of the decreased ELABELA level when combined with an elevated BNP level and LVEF ≥ 50% for HFpEF patients.Although it is clear that ventricular diastolic dysfunction plays a key role in HFpEF progression 59 , diastolic dysfunction is not synonymous with HFpEF but considered as a part of the normal again process 56 .In this case, the benchmarking to LVEF is not used for the diagnosis of HFpEF in a meaningful manner.The determination of the diagnosis value of plasma ELABELA on HFpEF may be compensate for the shortcomings of LVEF.In addition, multiple non-diastolic abnormalities including diabetes, obesity, chronic kidney disease, and hypertension are risk factors for HFpEF 56 .However, the occurrence of diabetes, obesity, chronic kidney disease, and hypertension was very low in the present cohort.This may be related to the specific subtypes of HFpEF.Indeed, a clinical phenotypic classification of HFpEF has already reported, mainly including (1) Vascular-related HFpEF; (2) Cardiomyopathy-related HFpEF; (3) Right heart-and pulmonary-related HFpEF; (4) Valvular-and rhythmrelated HFpEF; and (5) Extracardiac disease-related HFpEF 60 .Along this line, our data may indicate that the HFpEF patients in the cohort mainly belong to cardiomyopathy-related HFpEF or valvular-and rhythm-related HFpEF.Briefly, patients with HFpEF in the present cohort may be mainly caused by valvular heart disease and old myocardial infarction.
The various bioeffects of ELABELA or Apelin display important roles in HF development.We found that plasma ELABELA levels rather than Apelin levels were positively correlated with diastolic blood pressure and negatively correlated with plasma creatine and urea nitrogen.Chronic kidney disease and hypertension are closely associated with HF and are known as independent risk factors for HF progression 39,40,61 .Hypertension or chronic kidney disease interacts with HF, which jointly deteriorates the patient's physical condition.Increasing animal studies have demonstrated that peripheral ELABELA administration exhibits an antagonistic actions on multiple cardiovascular-related diseases including hypertension 62,63 and kidney injury 64,65 .Moreover, the levels of plasma ELABELA were remarkably lower and significantly negatively correlated with albuminuria, systolic and diastolic blood pressure in patients with type 2 diabetes 38 or essential hypertension 37 .Therefore, reduced circulating ELABELA levels might cause the elevation of the incidence of HF progression through hypertension or renal dysfunction.Along this line, ELABELA may be a potential therapeutic target/drug for HF.This assumption may be supported by the evidence from multiple animal studies that have already demonstrated the protective actions of peripheral ELABELA application on cardiac injury including ischemia/reperfusion injury, oxidative stress injury, hypertensive injury, and myocardial infarction 18 .However, there is currently a lack of direct clinical evidence for the therapeutic efficacy of ELABELA for HF patients, which awaits future clinical evaluation.
NT-proBNP and BNP, known as diagnostic indicators of HF 66,67 , were associated with the severity and mortality of HF 68 and important predictors for adverse events in HF patients 69 .Plasma ELABELA was significantly negatively associated with plasma BNP, while plasma Apelin was markedly positively related to plasma BNP in the present study.The negative relationship between ELABELA and BNP suggested that ELABELA protects against HF, possibly via its positive inotropic effect and attenuating cardiac remodeling.While the positive correlation between Apelin and BNP may be a compensatory outcome of reduced plasma ELABELA that enhances inotropic action and abolishes cardiac remodeling, thus exerting an anti-HF effect.Along this line, we found that the correlations between plasma ELABELA and cardiac function-related parameters (LVEF, LAD, LVEDd, LVEDs, IVST, LVPW, and RVIDd assessed by electrocardiography) were opposite to that between plasma Apelin and the above indexes.Unfortunately, both BNP and LVEF have poor predictive power for HF due to their low specificity 6,7 .Our results indicated that decreased ELABELA level might be a novel promising screening indicator for HF.In the present study, multivariate linear regression analysis revealed that plasma BNP only exhibited a significant negative impact on plasma ELABELA levels but not plasma Apelin levels.Thus, declined ELABELA plasma levels might be an underlying risk factor for HF progression and a potential predictor of a worse prognosis for HF.This concept can be supported by the multiple protective effects of ELABELA, including antihypertensive, cardioprotective, and renoprotective effects 18,19 .Similar to previous reports 27,41 , compared to ELABELA, the LVEF had a lower diagnostic value for HF with a lower predictive sensitivity and a comparable predictive specificity.Notably, although the predictive sensitivity of plasma ELABELA for the diagnosis of HF is comparable to that of plasma Apelin, plasma ELABELA had a higher diagnostic value and predictive specificity than plasma Apelin.Therefore, ELABELA might be superior to Apelin and LVEF for the diagnosis and prognosis of HF.Combined assessment of BNP and ELABELA may provide potential benefits for the diagnosis of HF.
The present study has several limitations.First, the current study population included only subjects from a single center.Thus, our findings may not be suitable for all ordinary populations due to the sample selection bias, including population and region bias.However, our findings are at least applicable to Chinese patients.Second, the sample size was small, which may reduce the reliability of the subgroup analysis.Third, the data was only dependent on the ELABELA/Apelin ELISA assay, the method is limited by the specificity of the antibody used in the ELISA kit.Fourth, patients in HF and non-HF groups were included based on typical signs, symptoms, and plasma BNP concentrations, with a lacking of a healthy control group or an HF group of different origins, such as patients with ischemic HF.It is still unclear whether there are differences in the levels of plasma ELAB-ELA between healthy individuals and HF patients of matched age or HF of different origins.Lastly, follow-up studies were not conducted and outcome data are not available.The correlation between ELABELA and major outcomes including hospitalization, readmission, and all-cause mortality due to HF in our cohort is unclear.Therefore, future longitudinal multicenter clinical studies with a larger sample size and a healthy control cohort or an HF cohort of different origins are necessary to further verify the effectiveness of ELABELA in clinical diagnostic practice.

Figure 2 .
Figure 2. Receiver Operating Characteristic (ROC) curves of ELABELA, Apelin, and left ventricular ejection fraction (LVEF) levels for diagnostic value for heart failure.

Table 6 .
The demographic and baseline characteristics of the HF patients with low level and high level of